Systems and methods of voltage-gated ion channel assays

ABSTRACT

Systems and methods are provided for optically measuring ion concentrations in biological samples. The systems and methods employ polymer-based optical ion sensors that include ion-selective ionophores and a pH sensitive chromionophore. Electrodes are providing for electrically stimulating the biological samples.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to UnitedStates Provisional Application No. 60/838,647, entitled, “HighThroughput Optical Sensor Arrays for Drug Screening,” filed on Aug. 17,2006, the entirety of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Electrically stimulated voltage-gated ion channels control many of themost basic functions in the human body, including the contraction ofmuscle cells and the propagation of nervous system signals via muscleand nerve cells. Various compounds are known to interfere with theproper operation of these voltage-gated ion channels.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a cell assay system suitable forobserving the impact of agents on the functioning of voltage-gated ionchannels in electrically stimulated cells. In one embodiment the cellassay system includes a post that has a distal end sized forintroduction into a biological sample holder and a plurality ofelectrodes for generating an electric field between the electrodes whenintroduced into the biological sample holder. At least one of theelectrodes is coupled to the post. In addition, a polymer-based opticalion sensor is positioned proximate the distal end of the post. Theoptical ion sensor includes at least one ionophore for selectivelybinding a predetermined ion and one pH-sensitive chromionophore. Thebinding of the predetermined ion alters the pH of the optical ionsensor. The chromionophore optically indicates the concentration of thepredetermined ion based on the pH of the optical ion sensor and theresulting fluorescence of the chromionophore.

The optical ion sensor can take a number of forms. In one embodiment,the optical ion sensor is removably coupled to the post, for example, asa removable insert. Alternatively, the optical ion sensor can be coupledto the biological sample holder or it may be suspended in a fluid in thebiological sample holder. In still other embodiments, the optical ionsensor is a particle inside of a cell in the biological sample holder.The optical ion sensor can be introduced into the cell through a varietyof means including injection, endocytosis, or phagocytosis. In stillother implementations, one optical ion sensor is coupled to the post orotherwise maintained outside of a cell and a second optical ion sensoris introduced into the cell. In implementations with two optical ionsensors, the optodes may have ionophores selective for different targetions and/or they may have chromionophres that have distinct fluorescenceproperties so that the system can simultaneous indicate theconcentration of two different ions.

In various embodiments, the cell assay system also includes acontrollable voltage source for generating a voltage across theelectrodes. The voltage can be period or constant. In oneimplementation, the voltage source can generate a voltage high enough toelectroporate a cell. The controllable voltage source also may provide avoltage sufficient to activate an ion channel in a cell.

In one embodiment, the cell assay system includes a means forintroducing an agent into the biological sample holder, such as a holethrough which an agent may be dispensed, a pipette, or aelectromechanical dispenser. The electromechanical dispenser, in oneparticular implementation includes a solenoid.

In one embodiment, the electrodes are two parallel electrodes coupled toopposing portions of the post. Alternatively, the electrodes may becoaxial. In still other embodiments, at least one electrode is coupledto the biological sample holder. The electrodes may be transparent,reflective, or opaque. In one particular embodiment, the cell assaysystem includes two pairs of opposing electrodes, which areperpendicular to one another.

The cell assay system may also include a light source and a lightsensor. A computing device may include modules for controlling each. Thelight sensor detects and measures the fluorescence of the optical ionsensors. The computing device may also include an analysis module foranalyzing the output of the light sensor, a voltage control module forcontrolling the voltage source, and an agent introduction module forcontrolling the agent introduction means. The analysis module, in oneembodiment, compares the output of the light sensor before anintroduction of an agent into the biological sample holder to the outputof the light sensor after the introduction of the agent into thebiological sample holder.

In another aspect, the invention relates to a cell assay system thatincludes an array of posts having optical ion sensors, such as thosedescribed above, positioned proximate thereto. The optical ion sensorscorresponding to each posts may be the selective for the same ions orthe they may be selective for different ions. The cell assay system alsoincludes a plurality of electrode sets corresponding to the array ofposts. The electrode sets are configured to generate an electric fieldwhen introduced into a corresponding biological sample holder in anarray of biological sample holders. The array of biological sampleholders, in one embodiment is a standard 6-well, 12-well, 24-well,48-well, 96-well, 384-well, and a 1534-well plate.

In one embodiment, the cell assay system includes a computing deviceincluding an agent introduction control module for controlling theintroduction of an agent into at least one of the biological sampleholders. The agent introduction control module controls the introductionof a plurality of agents into respective ones of the biological sampleholders in the array of biological sample holders. The computing devicemay also include an analysis module for comparing the fluorescence ofthe plurality of optical ion sensors before an introduction of at leastone agent into at least one of the respective biological sample holdersto the fluorescence of the corresponding plurality of optical ionsensors after the introduction of the at least one agent into therespective biological sample holders.

An additional feature of the cell assay system is a robotics module forrobotically introducing the array of posts into the array of biologicalsample holders. The robotics module may also configured to introduce thearray of posts into a plurality of arrays of biological sample holdersin sequence.

In another aspect, the invention relates to a method of conducting abiological assay. The method includes introducing a polymer-basedoptical ion sensor into a biological sample holder. The optical ionsensor includes at least one ionophore for selectively binding apredetermined ion and one pH-sensitive chromionophore. The binding ofthe predetermined ion alters the pH of the optical ion sensor. Thechromionophore optically indicates the concentration of thepredetermined ion based on the pH of the optical ion sensor and theresulting fluorescence of the chromionophore. The method also includesgenerating an electric field across a cell in the biological sampleholder, and measuring an output of a light sensor monitoring afluorescence of the optical ion sensor in response to the generation ofthe electric field. The method, in one embodiment includes varying theelectric field. Additional features include introducing an agent intothe biological sample holder and detecting a change in the output of thelight sensor in response to the introduction of the agent.

In addition, based on a detected change, the method, in one embodiment,determines that the agent is toxic. For example, the method maydetermine whether the agent is a nerve or heart toxin. Alternatively,the method may determine that the agent is a candidate for treating acondition or illness. Diseases or conditions known to be related tovoltage-gated ion channels include, for example, Central core disease,Hyperkalemic periodic paralysis, paramyotonia congenita, andpotassium-aggravated myotonia, Hypokalemic periodic paralysis, Malignanthyperthermia, Myotonia congenita, Long Q-T syndrome, Generalizedepilepsy with febrile seizures, Episodic ataxia type 1, Hemiplegicmigraine and allelic ataxias, X-linked congenital stationary nightblindness, Human Bartter Syndrome, and Human X-Linked RecessiveNephrolithiasis. In another implementation, the method may identify theagent by comparing it to fluorescence fingerprints of known agents.

In a further aspect, the invention relates to another method ofconducting a biological assay in which an array of biological sampleholders are provided. The method includes introducing an array ofpolymer-based optical ion sensors, such as those described above, intobiological sample holders in the array of biological sample holders.Electric fields are generated across cells in the biological sampleholders in the array of biological sample holders. The output of a lightsensor monitoring the fluorescence is then measured to observe theresponse of the optical ion sensors to the electric fields. The method,in one embodiment, includes introducing a first agent into one of thebiological sample holders in the array of biological sample holders, andintroducing a second agent into a second of the biological sampleholders in the array of biological sample holders. The method may alsoinclude detecting a change in the output of the light sensor resultingfrom the introduction of the first and second agents. In still anotherembodiment, the invention includes providing a second array ofbiological sample holders and robotically introducing the array ofoptical ion sensors into the second array of biological sample holders.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood from the following illustrativedescription with reference to the following drawings.

FIG. 1 is a schematic diagram of a cell assay system according to anillustrative embodiment of the invention.

FIGS. 2A-2D are cross sections of various optical ion sensorarrangements suitable for use in various implementations of the cellassay system of FIG. 1, according to an illustrative embodiment of theinvention.

FIGS. 3A-3D illustrate alternative electrode arrangements suitable forvarious implementations of the cell assay system of FIG. 1, according toan illustrative embodiment of the invention.

FIG. 4A is a perspective view of an illustrative optical ion sensorsupport, suitable for use in the cell assay system of FIG. 1, accordingto an illustrative embodiment of the invention.

FIGS. 4B-4D are perspective views of components of the optical ionsensor support of FIG. 4A, according to an illustrative embodiment ofthe invention.

FIG. 5 is perspective view of an alternative optical ion sensor supportsuitable for use in the cell assay system of FIG. 1, according to anillustrative embodiment of the invention.

FIG. 6 is a perspective view of a optical ion sensor support/biologicalsample holder configuration suitable for use on a microscope stage,according to an illustrative embodiment of the invention.

FIG. 7 is a schematic diagram of an alternative cell assay system,according to an illustrative embodiment of the invention.

FIG. 8A is a perspective view of an optical ion sensor array suitablefor use in the cell assay system of FIG. 7, according to an illustrativeembodiment of the invention.

FIG. 8B is a perspective view of a optical ion sensor array andbiological sample holder arrangement, according to an illustrativeembodiment of the invention.

FIG. 9 is a flow chart of a method conducting a biological assay using acell assay system, such as the cell assay system of FIG. 7, according toan illustrative embodiment of the invention.

FIG. 10A-10D are graphs of experimental data derived using a potassiumion-selective optical ion sensor to monitor cardiac myocyte cells,according to an illustrative embodiment of the invention.

FIGS. 11A and 11B are graphs of experimental fluorescence data observedfrom sodium ion-selective optical ion sensor particles introduced intothe interior of HL-1 cardiac cells.

FIG. 12 is a flowchart of a method of identifying an unknown agent 1200,according to an illustrative embodiment of the invention.

DESCRIPTION OF CERTAIN ILLUSTRATIVE EMBODIMENTS

To provide an overall understanding of the invention, certainillustrative embodiments will now be described, including methods andsystems for optical evaluation of ion concentrations in biologicalsamples. However, it will be understood by one of ordinary skill in theart that the methods and systems described herein may be adapted andmodified as is appropriate for the application being addressed and thatthe systems and methods described herein may be employed in othersuitable applications, and that such other additions and modificationswill not depart from the scope hereof.

FIG. 1 is a schematic diagram of a cell assay system 100 according to anillustrative embodiment of the invention. The cell assay system 100includes an optical ion sensor support 102, a biological sample holder104, an excitation light source 106, a light sensor 108, a roboticsassembly 110, and a computing device 111.

The optical ion sensor support 102 supports an optical ion sensor 112for positioning in the biological sample holder 104. In the illustrativeembodiment, the optical ion sensor support 102 take the form of a post114 extending from a platform 116, which is coupled to the roboticsassembly 111. The post can be of any material which is compatible withthe optical ion sensor 112. In various implementations, the optical ionsensor 112 is adhered to the optical ion sensor support 102 bydepositing onto the distal end of the post 114 a solution of optical ionsensor matrices dissolved in a solvent, such as in a polar organicsolvent like Tetrahydrofuran (THF). In such implementations, the post ispreferably formed from a material resistant to the solvent. Materialsresistant to THF include, without limitation, 304 stainless steel, 316stainless steel, Acetal polymer (marketed as DELRIN™ by E. I. du Pont deNemours and Company), bronze, carbon graphite, carbon steel, ceramicAl203, a perfluoroelastomer compound, such as CHEMRAZ™ marketed byGreene, Tweed, epoxy, HOSTELRY C™ alloy (marketed by HaynesInternational, Inc.), KALES™ elastomer (marketed by DuPont PerformanceElastomers), polychlorotrifluoroethylene, NYLON™ (marketed by E. I. duPont de Nemours and Company), Polyetherether Ketone (PEEK),polyphenylene sulfide, and PTFE.

The optical ion sensor 112 includes a film including a suspension ofoptical ion sensor matrices. The optical ion sensor matrices, ingeneral, include an ionophore, an additive, and a chromionophoresuspended in a polymer phase, for example, of polyvinyl chloride (PVC).The polymer phase also includes a plasticizer such as DOS. An ionophoresubstance that allows targeted ions to move across or into a membrane.Preferably the ionophore is selected to be lipid soluble. In addition,the ionophore is preferably an electrically neutral compound that formsa complex with a target ion. The ionophore is optically inactive in thevisible spectrum and does not change absorbance or fluorescencedepending on its state of complexation.

A chromoionophore is an ionophore that changes its optical properties inthe visible spectrum depending on the state of complexation.Chromoionophores are preferably proton sensitive dyes that changeabsorbance (and fluorescence in many cases) depending on its degree ofhydrogen complexation (protonation). The chromionophores are preferablyhighly lipophilic to prevent the chromionophores from leaching out ofthe optical ion sensor matrix. Suitable chromionophores includeChromoionophore II and Chromionophore III. Chromionophore II exhibitslight absorbance peaks at 520 nm and 660 nm and a fluorescent emissionpeak at 660 nm. Chromionophore III has light absorbance peaks at 500 nmand 650 nm and fluorescent emission peaks at 570 nm and 670 nm.

For optical ion sensors targeting cations, the additive can be any inertlipophilic component that has a negative charge associated with it. Foroptical ion sensors targeting anions, the additive is positivelycharged. The purpose of the additive is to imbed charge sites within thepolymer phase, to help enforce charge neutrality within the optical ionsensor 112. The additive allows the polymer phase to carry an equalamount of charged particles as additive. The concentration ratio ofadditive to chromoionophore is preferably 1:1, thereby allowing thechromoionphore to become completely protonated or de-protonated. Onesuitable additive for optical ion sensors targeting negative ions ispotassium Tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTFPB). Thelipophilic anionic component TFPB-molecules are retained by the polymerphase and the potassium ions are either complexed by the ionophore, orexpelled into the sample solution through diffusion. In one particularimplementation, the optical ion sensor film is composed of a suspensionproduced from about 60 mg of DOS, 30 mg of PVC, and up to about 5 mg, ofadditive, ionophore, and chromionophore.

Once the above components are dissolved into the polymer phase to formthe optical ion sensor 112 and are exposed to a sample solution, theoptical ion sensor 112 becomes active. It now continuously extracts orexpels analyte cations (system can work with anions as well using ioncoextraction) depending on ion activity in the sample solution. With a1:1 additive-chromoionophore ratio, with zero target ions present in thesample solution, the optical ion sensor 112 remains completelyprotonated to achieve charge neutrality. As the target ion concentrationincreases, the ionophores in the optical ion sensor 112 extract thetarget ions into the optical ion sensor 112. To maintain chargeneutrality of the optical ion sensor 112, hydrogen ions are strippedfrom the chromoionphores in the optical ion sensor 112 and expelled intothe sample solution. The expelling of hydrogen ions alters the pH of theoptical ion sensor 112, thereby altering its fluorescent properties. Todetect analyte anions (for example, chloride or nitrite ions), theoptical ion sensor uses ion-coextraction, as opposed to protonexpulsion. To detect neutral analytes, an additional agent known tointeract with the target analyte to yield an ion is added to thebiological sample holder 104. An ionophore is then selected to detectthe resultant ion.

The following is a non-limiting, illustrative list of targetion/ionophore pairings suitable for use in the optical ion sensors:Potassium/Potassium Ionophore III (BME-44), Sodium/Sodium Ionophore IV,Sodium/Sodium Ionophore V, Sodium/Sodium Ionophore VI, Calcium/CalciumIonophore III, and Calcium/Calcium ionophore IV. For target anions,illustrative target ion/ionophore pairings include chloride/ChlorideIonophore III and nitrite/Nitrite Ionophore I.

The film of the optical ion sensor can be produced in various ways. Inone implementation, as described above, a predetermined amount of theoptical ion sensor suspension (i.e., the combined polymer phase,ionophore, additive, and chromionophore) is dissolved in a solvent, suchas THF. The solution is then deposited, sprayed, or spun onto a surface.The solvent then evaporates leaving the optical ion sensor film on thesurface.

In another implementation, the film is formed form a deposition ofoptical ion sensor microspheres. To produce the microspheres, an opticalion sensor emulsion is formed by injecting an optical ion sensorsuspension dissolved in THF (e.g., 16 mL THF/100 mg PVC) into a pHbuffered solution. The optical ion sensor suspension includesapproximately 60 mg of DOS, 30 mg of PVC, and up to approximately 5 mgof chromionophore, additive, and ionophore. The emulsion is thensubmerged in a sonicating water bath. Typically, 50 μL of the opticalion sensor suspension/THF solution is injected into 1,000-1,500 μL ofbuffered solution. The resulting emulsion contains a mixture ofspherical optical ion sensor particles ranging in size from 200nanometers to 20 microns. The resulting emulsion can be spun, sprayed,or evaporated onto any surface to create a porous optical ion sensormembrane. Films formed from microspheres tend to expose a greatersurface area of optical ion sensor to a given sample, yielding improvedperformance characteristics.

The optical ion sensor support 102 also supports a pair of electrodes117, which are coupled to opposite sides of the support 102. Theelectrodes 117 are also preferably THF resistant and can be made of, forexample, and without limitation, platinum or silver chloride.Alternative electrode configurations are described below in relation toFIGS. 3A-D.

The optical ion sensor support 102 includes an agent introduction means118. The agent introduction means 118 can include a channel boredthrough the post 114, a pipette, or an electromechanical dispenserdevice, such as a solenoid or electrostatically driven plunger orsyringe. The pipette or electromechanical dispenser may be positionedwithin a borehole formed in the post 114, or it may be coupled to theplatform 116. The agent introduction means 118 allows a user tointroduce an agent into the biological sample holder 104. In alternativeimplementations, the agent introductions means 118 can be attached tothe platform 116. The agent introduction means 118 may be coupled to anagent reservoir 120 which stores the agent to be introduced. Inalternative implementations, the optical ion sensor support 102 includestwo or more agent introduction means 118. In one such implementation, afirst agent introduction means 118 is used to introduce a therapeutic orother biologic into the biological sample holder to assay its effect onthe cells located therein. The additional agent introduction means 118may be used for introducing other therapeutics or biologics or tointroduce optical ion sensor particles, described further below, foruptake, for example, into the cells in the biological sample holder 104.

The electrodes 117 coupled to the optical ion sensor support 102 areenergized by a voltage source 122. The voltage source 122 may provide anAC and/or DC voltage to the electrodes 117 for generating an electricfield between the electrodes 117. The voltage source 122, in oneimplementation is capable of providing a sufficient voltage toelectroporate cells 124 in the biological sample holder. The voltagesource 122 can also provide generate smaller magnitude voltages that aresufficient to activate voltage-gated ion channels in the cells 124, butnot to cause electroporation.

The biological sample holder 104 holds a biological sample for analysisby the cell assay system 100. The biological sample can include, asillustrated in FIG. 1, cells 124 adhered to the walls of the biologicalsample holder 104, for example, in a monolayer, or cells 124 suspendedin a liquid buffer. The biological sample holder 104 is preferablytransparent, or at least includes a transparent region through which theoptical ion sensor 112 can be excited and through which the results ofsuch excitement can be monitored.

The optical ion sensor 112 is illuminated with a light source 106 toexcite the chromionophores suspended therein. The light sourcepreferably can be tuned to generate one or more predeterminedwavelengths of light, preferably in the visible portion of theelectromagnetic spectrum, selected to excite the particularchromionophore used in the optical ion sensor 112. Alternatively, thelight source may generate a wide spectrum light. In one implementation,the light source 106 is coupled to the optical ion sensor support 102.

The fluorescence of the optical ion sensor 112 is detected by a lightsensor 108. The light sensor 108 may include a charge coupled device, afluorometer, a photomultiplier tube, or other suitable device formeasuring fluorescence. In one implementation, a spectrophotofluorometeris used to satisfy the roles of the light source 106 and the lightsensor 108. The light sensor 108 may also be coupled to the optical ionsensor support 102.

A robotics assembly 110 controls the position of the optical ion sensorsupport 102 and/or the biological sample holder 104 to control theintroduction of the optical ion sensor 112 into the biological sampleholder. In the illustrative embodiment, the robotics assembly 110includes a robotic arm 124 coupled to the platform 116 of the opticalion sensor support 102 for raising and lowering the optical ion sensorsupport 102 into and out of the biological sample holder. The roboticarm 124 may also provide three dimensional movement control of theoptical ion sensor support 102, for example, to move the optical ionsensor support 102 between different biological sample holders 104, forexample, arranged as multiple wells in a standard multi-well plate. Therobotics assembly may also move the optical ion sensor support 102between a biological holder sample 104 and a preparation bath used toprotonate the optical ion sensor film 112 and to strip the optical ionsensor 112 of target ions previously brought into the optical ion sensor112.

The computing device 111 controls the various components of the cellassay system 100. The computing device 111 may be a single computingdevice or multiple computing devices providing the variousfunctionalities used to control the cell assay system. Thesefunctionalities are provided by an excitation control module 126, avoltage control module 128, an agent introduction module 130, a roboticsmodule 132, and an analysis module 134. The excitation control module126 controls the light source 108 to emit one or wavelengths ofexcitation light. The voltage control module 128 controls the voltagesource 122 to generate a constant or time-varying electric field betweenthe electrodes 117. The agent introduction module 130 controls theintroduction of an agent into the biological sample holder 104 via theagent introduction means 118. The robotics module 134 controls therobotics assembly 110. The analysis module 134 analyzes the output ofthe light sensor 108. For example, the analysis module 134 may comparethe output of the light sensor 108 to determine the response of applyingvarious voltages across the electrodes 117. The analysis module may alsoanalyze the output of the light sensor 108 before and after an agent isintroduced into the biological sample holder 104 to determine the effectof the agent on the cells 124 in the biological sample holder 104. Theanalysis module 134 may also control the other modules in the computingdevice, i.e., the excitation control module 126, the voltage controlmodule 128, the agent introduction module 130, and the robotics module132, to coordinate an assay protocol. The computing device 111 and/ordevices may also include various user interface components, such as akeyboard, mouse, trackball, printer, and display.

A module may be implemented as a hardware circuit comprising custom VLSIcircuits or gate arrays, off-the-shelf semiconductors such as logicchips, transistors, or other discrete components. A module may also beimplemented in programmable hardware devices such as field programmablegate arrays, programmable array logic, programmable logic devices or thelike.

Modules may also be implemented in software for execution by varioustypes of processors. An identified module of executable code may, forinstance, comprise one or more physical or logical blocks of computerinstructions which may, for instance, be organized as an object,procedure, or function. Nevertheless, the executables of an identifiedmodule need not be physically located together, but may comprisedisparate instructions stored in different locations which, when joinedlogically together, comprise the module and achieve the stated purposefor the module. A module of executable code could be a singleinstruction, or many instructions, and may even be distributed overseveral different code segments, among different programs, and acrossseveral memory devices.

The various modules are in communication with the various devices theycontrol or obtain data from. They maybe connected over a local areanetwork, wirelessly, over a bus, or over typical cables known in the artof computer interfaces for connecting computing devices withperipherals.

FIGS. 2A-2D are cross sections of various optical ion sensorarrangements suitable for use in various implementations of the cellassay system 100 of FIG. 1. FIG. 2A is a cross section of a firstoptical ion sensor arrangement 200 suitable for use in the cell assaysystem of FIG. 1. The optical ion sensor arrangement 200 includes anoptical ion sensor support 202 and a biological sample holder 204. Thebiological sample holder 204 includes a monolayer of cells 206 adheredto the biological sample holder 204. Alternatively, the biologicalsample holder 204 holds cells suspended in a buffer. The optical ionsensor support 202 and biological sample holder 204 correspond to theoptical ion sensor support 102 and biological sample holder 104 ofFIG. 1. Electrodes 208 are coupled to opposing sides of the optical ionsensor support 202. A optical ion sensor film 210 is coupled to thedistal end of the optical ion sensor support 202.

FIG. 2B is an alternative optical ion sensor arrangement 250 for use inan alternative implementation of the cell assay system 100 of FIG. 1.The optical ion sensor arrangement 250 includes an electrode support 252and a biological sample holder 254. Electrodes 255 are coupled toopposing sides of the electrode support 252. The biological sampleholder 254 includes a monolayer of cells 256 adhered to the surfaces ofthe biological sample holder 254 or cells suspended in a buffer. Insteadof including an optical ion sensor film adhered to a support, theoptical ion sensor arrangement 250 relies upon optical ion sensorparticles 258 introduced into the cells 256 adhered to the biologicalsample holder 254.

To introduce optical ion sensors into cells, the optical ion sensors areproduced as optical ion sensor particles 258. The optical ion sensorparticles 258 are fabricated in a similar fashion as the optical ionsensor film 112 described above. One such particle 258, the optical ionsensor nanosphere, is produced according to the following procedure.First a optical ion sensor suspension is dissolved in 500 μl of THF. Thesuspension preferably includes 60 mg of DOS, 30 mg of PVC and up toabout 5 mg of chromoionophore, ionophore, and additive to form an optodesolution. Then, 500 μl of CH₂Cl₂ is added to bring the total volume to 1ml. Next, a PEG-lipid solution is prepared by adding dissolving aPEG-lipid into 5 ml of a water, salt and buffer solution. A TAT peptidecan be added to the PEG-lipid via an amine linkage to aid the resultingnanospheres in entering cells.

The nanospheres are formed by adding 100 μl of optode solution drop wiseto 5 ml of the PEG-lipid solution while the solution is being sonicatedby a probe tip sonicator. Additional sonication is performed forapproximately 2-3 minutes. The resultant nanosphere solution is sprayedthrough a nitrogen feed air gun into a beaker several times to removeexcess solvent. If desired, the nanosphere solution is pushed through a0.22 μm filter to remove the larger spheres.

The optical ion sensor particles 258 are introduced into the cells 256in one of two ways. In one method, the optical ion sensor particles 258are introduced into a buffer liquid deposited in the biological sampleholder 254. A voltage source then generates a voltage across theelectrodes 255 sufficiently strong to electroporate the cells 256,thereby allowing the optical ion sensor particles 258 to enter directlyinto the cells. In the other method, the surfaces of the optical ionsensor particles 258 are first coated with a substance, for exampletransferrin or folate, which aid in the optical ion sensor particles 258crossing over cell membranes. The optical ion sensor particles 258 areintroduced into a buffer in the biological sample holder 254. The cells256 bring the optical ion sensor particles 258 into their interior invesicles via endocytosis, pinocytosis, or phagocytosis, or similarbiological processes. The substance applied to the optical ion sensorparticles 258 breaks down the vesicle membrane, releasing the opticalion sensor particles 258 into the cell cytoplasm.

FIG. 2C is a second alternative optical ion sensor arrangement 270 foruse in an alternative implementation of the cell assay system 100 ofFIG. 1. The optical ion sensor arrangement 270 includes an optical ionsensor support 272 and a biological sample holder 274. Electrodes 275are coupled to opposing sides of the optical ion sensor support 272. Anoptical ion sensor film 276 is coupled to the distal end of the opticalion sensor support 272. A cell monolayer 278 adheres to the surfaces ofthe biological sample holder 274. Alternatively, cells are suspended ina buffer. In addition, optical ion sensor particles 280 are introducedinto the cells of the cell monolayer 278. Preferably the chromionophoresused in the optical ion sensor film 276 differ from the chromionophoresused in the optical ion sensor particles 280. In particular, thedifferent chromionophores preferably have distinguishable fluorescencecharacteristics such that an analysis module analyzing the output of alight sensor monitoring the optical ion sensor arrangement 270 candifferentiate between the output of the optical ion sensor film 272 andthe optical ion sensor particles 280. As a result, the analysis modulecan differentiate between intracellular target ion concentration andextracellular target ion concentration. In addition, the optical ionsensor film 272 may include different ionophores than those included inthe optical ion sensor particles 280. Thus, the optical ion sensorarrangement 270 can monitor the concentrations of two different targetions.

FIG. 2D is a third alternative optical ion sensor arrangement 290 foruse in an alternative implementation of the cell assay system 100 ofFIG. 1. The optical ion sensor arrangement 290 includes an electrodesupport 292 and a biological sample holder 294. The biological sampleholder 294, in addition to a cell monolayer 296 or cells suspended in abuffer, includes a removable optical ion sensor film 298. The removableoptical ion sensor film 298, for example, can be a glass cover slip orother transparent surface coated with an optical ion sensor film.

FIGS. 3A-3D illustrate alternative electrode arrangements suitable forvarious implementations of the cell assay system 100 of FIG. 1. FIG. 3Ais a cross section of a first alternative electrode arrangement 300. Theelectrode arrangement 300 includes an electrode support 302 and abiological sample holder 304. The electrode support 302 includes a firstelectrode 306 coupled to the distal end of the electrode support. Asecond electrode 308 is coupled to the bottom of the biological sampleholder 304. The second electrode 308 is preferably formed from atransparent conductor, such as indium tin oxide. The biological sampleholder 304 includes a monolayer of cells 310 adhered to the surface ofthe second electrode 308. Alternatively, the cells 310 may be suspendedin a fluid in the biological sample holder 310.

FIG. 3B is a cross section of a second alternative electrode arrangement320, according to an illustrative embodiment of the invention. Theelectrode arrangement 320 includes and optical ion sensor support 322and a biological sample holder 324. The optical ion sensor support 322includes an optical ion sensor film 326 coupled to its distal end.Electrodes 328 are coupled to opposing sides of the biological sampleholder 324.

FIG. 3C is perspective view of a third alternative electrode arrangement350, according to an illustrative embodiment of the invention. In thethird electrode arrangement 350, electrodes 352 and 354 are arrangedcoaxially, with one electrode 352 filling a cavity in the interior of anelectrode support 356, which can be positioned in a biological sampleholder. The second electrode 354 surrounds the exterior of the electrodesupport 356.

FIG. 3D is perspective view of a fourth alternative electrodearrangement 370, according to an illustrative embodiment of theinvention. This electrode arrangement 370 includes two pairs ofelectrodes 372 and 374 coupled to an optical ion sensor film 376. Eachpair of electrodes 372 and 374 can be individually energized to generateperpendicular electrical fields across cells in a biological sampleholder.

FIG. 4A is a perspective view of an illustrative optical ion sensorsupport 400, suitable for use in the cell assay system 100 of FIG. 1.The optical ion sensor support 400 includes three constituentcomponents, a housing 402, an optical ion sensor insert 404, and aplatform 406, described in further detail in FIGS. 4B-4D.

FIG. 4B is a perspective view of the housing 402 of the optical ionsensor support 400. The housing 402 includes a post 408 sized forinsertion into a biological sample holder. Electrodes 410 are formed inthe post. In one implementation, the distance between the platform 406and the housing 402 can be changed to adjust the distance between cellsin a biological sample holder and the distal end of the optical ionsensor insert 404. The housing 402 includes two dosing holes 412 forintroducing agents into a biological sample holder. The housing 402 alsoincludes an pair of mounting holes 414 for coupling the housing 402 tothe platform 406, and, in some implementations, through the platform 406to a robotic arm. The mounting holes 414 may be threaded, for example,for acceptance of bolts to couple the components together. Adjustment ofsuch bolts, in one implementation can be used to adjust the distance ofthe distal end of the optical ion sensor insert 404 to cells in abiological sample holder.

FIG. 4C is a perspective view of the optical ion sensor insert 404,according to an illustrative embodiment of the invention. The opticalion sensor insert 404 is constructed of DERLIN™ Acetal polymer and isshaped to fill a cavity milled into the housing 402. The optical ionsensor insert 404 can be removably inserted into the housing 402 suchthat it its distal end aligns with the bottom surface of the housing402. A optical ion sensor film 420 is coupled to the distal end of theoptical ion sensor insert 404. During operation, various optical ionsensor inserts 404 incorporating different ionophores in their opticalion sensor films 420 can be alternately inserted and removed into thehousing 402 to measure the varying concentrations of corresponding ions.

FIG. 4D is a perspective view of the platform 406 used in the opticalion sensor support 400. The platform includes a cavity 430 through whichthe optical ion sensor insert 404 can be inserted and withdrawn, alongwith dosing holes 432 and mounting holes 434 which align with the dosingholes 412 and mounting holes 414 of the housing 402.

FIG. 5 is perspective view of an alternative optical ion sensor support500 suitable for use in the cell assay system 100 of FIG. 1. The opticalion sensor support 500 includes a body 502 and a platform 504 coupledtogether at a hinge 506. The body 502 includes two electrodes 508 andtwo optical ion sensor films 510 and 512. The optical ion sensor films510 and 512 may include ionophores that are selective for the same ion.Alternatively, the optical ion sensor films 510 and 512 includeionophores that are selective for different ions. In such embodiments,the optical ion sensor films 510 and 512 also include differentchromionophores whose fluorescence characteristics can be distinguishedfrom one another. In one embodiment the hinge 506 keeps the body 502 atan angle with respect to the horizontal to reduce the likelihood ofbubble formation at the site of the electrodes 508. In otherembodiments, the optical ion sensor films 510 and 512 are disposed onthe distal ends of removal optical ion sensor inserts, such as theoptical ion sensor inserts 404 of FIG. 4A. The hinge 506, in thisembodiment, allows the body 502 to rotate with respect to the platform504 such that the optical ion sensor inserts are accessible forinsertion and removal.

FIG. 6 is a perspective view of a optical ion sensor support/biologicalsample holder configuration 600 suitable for use on a microscope stage.This configuration includes a base plate 602 coupled to a biologicalsample holder 604 at a hinge 606. The base plate 602 includes a bath608. Electrodes 610 are coupled to at least two of the four opposingwalls of the bath. A optical ion sensor film 612 is disposed in thebottom of the bath 608, for example, on a glass cover slip. Thebiological sample holder 604 includes a clamp to accept a transparentsubstrate with a cell monolayer formed thereon. The biological sampleholder includes an optical through hole 614 allowing the fluorescence ofthe optical ion sensor film 612 to be viewed through the top of thebiological sample holder 604 or through the bottom of the base plate602. In one mode of operation, the base plate 602 is placed on the stageof a microscope. An optical ion sensor film 612 selective for a desiredion is placed within the bath 608, and the biological sample holder 604,holding a sample, is lowered into position. The resulting fluorescencecan be monitored through the optical through hole 612 by a viewer viathe microscope. Alternatively, a camera can be attached to themicroscope to record the resultant fluorescence.

FIG. 7 is a schematic diagram of an alternative cell assay system 700.The cell assay system 700 is similar to the cell assay system 100 ofFIG. 1. However, the cell assay system 700 includes an array 701 ofoptical ion sensor supports 702. The optical ion supports 702 are sizedand spaced in the array 701 such that the optical ion sensor supports702 can be simultaneously introduced into multiple correspondingbiological sample holders 704. The optical ion sensor supports 702protrude from a platform 703. The array may be one-dimensional or twodimensional. The biological sample holders 704, in variousimplementations, are wells in a standard 6-, 12-, 24-, 48-, 96-, 384-,or 1534-well plate. The array 701 may include a sufficient number ofoptical ion sensor supports 702 to analyze an entire plate at once, orit may include a smaller number of optical ion sensor supports 702,requiring the array 701 be repositioned one or more times for each plateof biological sample holders 704 provided for analysis. Each optical ionsensor support 702 includes a corresponding set of electrodes 706, anoptical ion sensor 708, and an agent introduction means 710. Theelectrodes can take the form of any of the electrode arrangementsdescribed above in FIG. 1, FIGS. 3A-3D, FIG. 4, FIG. 5, and FIG. 6.

The optical ions sensor 708 for each optical ion sensor support 702 canbe arranged according to any of the optical ion sensor arrangementsdescribed above in FIG. 1, FIGS. 2A-2D, FIG. 4, FIG. 5, and FIG. 6. Inone implementation, each optical ion sensor support 702 incorporates thesame optical ion sensor 708. In alternative implementations, the opticalion sensor varies in each row or column of optical ion sensor supports702 of the array 701. In still other implementations, the optical ionsensor 708 selected for each optical ion sensor support 702 is chosenindividually or in a group-wise fashion.

The agent introduction means 710 corresponding to each optical ionsensor support 702, in one implementation, includes passive dosage holesfor administering agents. In alternative implementations, the agentintroduction means 710 includes a solenoid or electrostatically drivenmechanisms for introducing agents into biological sample holders 704. Instill another implementation, the agent introduction means 710 for eachoptical ion sensor support 702 includes a pipette. The agentintroduction means 710 are preferably coupled to an agent reservoir 712.As depicted in FIG. 7, the agent included in each agent reservoir 712may vary for each optical ion sensor support 702 or by row or column ofoptical ion sensor supports 702 in the array 701.

The cell assay system 700 includes at least one light source 714 forexciting the optical ion sensors 708 associated with each optical ionsensor support 702. In an alternative implementation, for example, eachrow of optical ion sensor supports 702 includes a different optical ionsensor 708. In this embodiment, the cell assay system 700 includes aseparate light source 714 for each row tuned to the wavelength of theoptical ion sensors 708 in that row. Alternatively, the cell assaysystem 700 may have separate light sources 714 for each optical ionsensor 708 or group of optical ion sensors 708.

Similarly, the cell assay system includes at least one light sensor 716.As with the light sources 714, the cell assay system may have a singlelight sensor 716 for the entire array, one light sensor 716 per row orcolumn of optical ion sensor supports 702, or one light sensor 716 foreach individual optical ion sensor 708 or group of optical ion sensors708.

The cell assay system includes a voltage source 718 for generatingvoltages across the sets of electrodes 706. The voltage source 718 canconnect to electrical interconnects integrated into the platform 703 ofthe array 701. In addition, the voltage source 718 can itself beincorporated into the platform 703. The voltage source 718 can providean AC and/or a DC voltage.

The cell assay system 700 also includes a robotics assembly 720. Therobotics assembly 720 can control the position of the platform in threedimensions using a robotic arm 722. The robotics assembly 720 can alsomaneuver multi-well plates into and out of position beneath the array701 so that the array 701 can be used in high throughput assays,analyzing samples in multiple plates in series.

The various components described above are controlled and monitored by acomputing device 724 similar to the computing device 111 of FIG. 1. Thecomputing device 724 includes an agent introduction module 726 forcontrolling the agent introduction means 710, a voltage control module728 for controlling the voltage source 718, a excitation control module730 for controlling the light source(s) 714 included in the cell assaysystem, a robotics module 732 for controlling the robotics assembly 720,and an analysis module 734 for directing assay protocols and analyzingthe output of the light sensor(s) 716 included in the cell assay system700. For example, the analysis module 734 determines the concentrationsof ions in the biological sample holders 704 over time. The analysismodule 734 also compares the data collected for each biological sampleholder 704 for comparative analysis. The comparative analysis may takeinto account knowledge of the various agents introduced into eachrespective biological sample holder 704.

FIG. 8A is a perspective view of an optical ion sensor array 800suitable for use as the array 701 in the cell assay system 700. Theoptical ion sensor array 800 includes 48 optical ion sensor supportposts 802 protruding from a platform 804. Each optical ion sensorsupport post 802 is sized to fit into a well of a standard 48- or96-well assay plate. The distal end of each ion support post 802 couplesto an optical ion sensor 806. The optical ion sensors 806 coupled to theoptical ion sensor support posts 802 may vary by row, by column, or byindividual or group of optical ion sensor support posts 802. Silverchloride electrodes 808 couple to opposing sides of the optical ionsensor support posts 802. The electrodes 808 electrically connect tointerconnects in the interior of the platform 804. The interconnectsconnect to a voltage source via a series of pins 810 extending from oneside of the platform 804.

FIG. 8B is a perspective view of a optical ion sensor array/biologicalsample holder arrangement 850. The arrangement 850 includes the array800 of FIG. 8A and a 96-well plate biological sample holder 852. Inoperation, the array 800 is lowered into a first half of the biologicalsample holder 852 for a first assay. It is then lowered into the secondhalf of the biological sample holder 852 for a second assay.

FIG. 9 is a flow chart of a method conducting a biological assay 900using a cell assay system, such as the cell assay system 700 of FIG. 7.The following methodology is particularly suited for analyzing theimpact of various agents on cellular voltage-gated ion channels,including potassium channels, sodium channels, and calcium channels. Themethod begins with providing a plurality of biological sample holdersholding biological samples (step 902). The biological samples, in oneimplementation include cells either adhered to the walls of thebiological sample holders covered with a buffer solution, or cellssuspended in a buffer solution. Though any type of cell can be monitoredusing the cell assay system, cells of particular interest include nervecells or muscle cells in which voltage-gated ion channels regulate thepropagation of action potentials along cell membranes and between cells.Other cells of interest include cells with non-wild-type ion channels,for example, cells with overactive or underactive voltage-gated ionchannels.

Next, optical ion sensors are introduced into the biological sampleholders (step 904). In one implementation, the optical ion sensors areintroduced into the buffer surrounding the cells by lowering optical ionsensor support posts into the biological sample holders. In thealternative, or in addition, optical ion sensor particles are introducedinto the cells in the biological sample holders. As described above,optical ion sensor particles can be introduced either by electroporatingthe cells via electrodes positioned in the biological sample holders orby the chemistry applied to the optical ion sensor particles breachingvesicle membranes within the cells. Similarly, the optical ion sensorsensors can be introduced into the cells using pico-injection, beadloading, a gene gun, or through liposomal delivery techniques known inthe art. As described above the optical ion sensors include at least oneionophore for selectively binding a predetermined ion thereby altering apH of a optical ion sensor and one pH-sensitive chromionophore foroptically indicating the concentration of an ion in a fluid surroundingthe optical ion sensor corresponding to the ionophore. Ion concentrationis indicated by the pH of the optical ion sensor and the resultingfluorescence of the chromionophore.

Sets of electrodes then apply a time-varying voltage across the cells inthe biological sample holders (step 906). Preferably at least one of theelectrodes in each set is coupled to an optical ion sensor support postwith which an optical ion sensor is introduced into a particularbiological sample holder.

At step 908, an agent, such as a therapeutic, toxin, biologicalmacromolecule (such as a nucleic acid, an antibody, a protein or portionthereof, e.g., a peptide), small molecule (of 2000 amu or less, 1000 amuor less or 500 amu or less), protein, virus, bacteria, chemicalcompound, mixture of chemical compounds, or an extract made frombiological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues, or other biologically activeagent is introduced into one or more of the biological sample holders.In one particular implementation using an array of biological sampleholders, no agent is introduced into a first row of biological sampleholders to preserve a control. A first agent is introduced into a secondrow of biological sample holders. Additional agents are added toadditional rows of the array of biological sample holders.

At step 910, the fluorescence of the optical ion sensors introduced intothe biological sample holders is monitored. The monitoring preferablybegins prior to introduction of the agents at step 908 and continuesthereafter. Changes in ion concentration resulting from the applicationof the voltage and/or the introduced agents are then determined based onthe monitoring (step 912). By comparing the changes in ion concentrationafter adding an agent, one can determine whether an agent is toxic tothe cells being tested. For example, if the ion concentration variesperiodically in relation to an electrical stimulus prior to addition ofan agent, and the addition of the agent results in a substantial changeto the periodicity or amplitude of the concentration variation, one canconsider the agent toxic. Alternatively, if such a change is in factdesired, for example, to treat a disease or condition, the agent mightbe considered a candidate therapeutic for further testing. The impact ofthe agent on ion flux can be compared to control agents with knownimpacts on voltage-gated ion channels. In one implementation, a libraryof agents may be tested in such a fashion as a high throughput screen toselect candidate therapeutics and/or to rule out agents in the librarythat cause undesirable effects on voltage-gated ion channels.

EXAMPLE 1

In one particular example, a cell assay system built in accordance withprinciples of the invention can be used to conduct hERG screeningassays. The hERG gene regulates the activity of potassium ion channelsin cardiac myocyte cells. In a functioning heart, electrical voltagesare generated by modified myocytes in the sinoatrial andatrioventricular nodes (pacemaking nodes). Such voltages are propagatedthrough the heart by the action of various voltage-gated ion channelsthat alter the voltage gradients across cardiac cell membranes byexchanging sodium, calcium, and potassium ions across the cell membrane.Various agents are known to interfere with the hERG gene, or otherwiseblock potassium channel operation, resulting in arrhythmia andpotentially heart failure. Thus, when testing various agents fortoxicity, one useful test includes testing for interference with cardiaccell potassium channels.

To test for potassium channel interference, cardiac myocyte cells areprovided in a monolayer adhered to a biological sample holder. Apotassium selective optical ion sensor is introduced into the biologicalsample holder. Electrodes coupled to the optical ion sensor replicatethe pacemaking functionality of the sinoatrial and atrioventricularnodes. As potassium ions are passed into and out of the myocytes bypotassium channels, the fluorescence of the potassium selective opticalion sensor varies in a detectable fashion.

FIGS. 10A and 10B are graphs charting the intensity of potassiumion-selective optical ion sensor fluorescence monitoring the activity ofmyocyte cells in a biological sample holder. Voltage applied acrosselectrodes in the biological sample holder regulates the pulse rate ofthe myocytes to 1.0 Hz. The graph of FIG. 10A includes normalized rawfluorescence measurements, both observed and filtered. The graph of FIG.10B illustrates a Fast Fourier Transform breakdown of the raw data,demonstrating the fluorescence intensities by frequency. As would beexpected, in the chart of 10B, a spike in intensity is visible atapproximately 1.0 Hz, the pulse of the cells.

FIGS. 10C and 10D are graphs charting intensity of potassiumion-selective optical ion sensor fluorescence the after 2.0 μM ofHaloperidol, a known potassium channel blocker, was introduced into thebiological sample holder holding the myocyte cells. The intensity offluorescence oscillations at 1.0 Hz, the pulse of the myocyte cells, isreduced by 30% in FIG. 10D in comparison to FIG. 10B, providing evidenceof the incapacitation of potassium channels. That is, with each beat,the change of potassium concentrations in the environment outside of themyocyte cells exposed to Haloperidol is 30% less than observed withcells not exposed to the agent.

EXAMPLE 2

FIGS. 11A and 11B are charts depicting fluorescence data observed fromsodium ion selective optical ion sensor particles introduced into theinterior of HL-1 cardiac cells. Electrodes energized at a frequency of1.0 Hz stimulate the cells. As the cells beat, sodium channels in thecells open and close, allowing sodium ions to move in and out of thecells. As the sodium concentration in the intracellular environmentchanges, the fluorescence of the optical ion sensor changes. FIG. 11Aillustrates the averaged raw and filtered normalized fluorescence dataindicative of sodium concentration in the HL-1 cells. FIG. 11Billustrates the fluorescence oscillation amplitude by frequency.

The optical ion sensors used to obtain the experimental data for FIGS.10A-10D and FIGS. 11A and 11B fluoresce primarily at a wavelength of 670nm. To simultaneously observe extracellular potassium and intracellularsodium, the potassium-selective optical ion sensors can be formulatedwith chromionophores that fluoresce at a different wavelength than thechromionophores used in the sodium-selective optical ion sensors.Similar evaluations can be carried out to determine the effect ofvarious agents on the calcium and sodium channels of neurons.

ADDITIONAL APPLICATIONS

The Examples described above relate primarily to using electrodes topace electrically excitable cells and observing the resulting activityof voltage-gated ion channels in those cells. The systems and methodsdescribed above can also be used to evaluate the activity ofvoltage-gated ion channels in cells which are not paced. For example,the electrodes, in one implementation can apply a DC voltage to cells ina biological sample holder to force and hold a voltage-gated ion channelinto a desired state. The systems described above can then be used toanalyze the effect of various agents on the activity of those ionchannels. For example, the systems can detect whether the agents preventthe ion channel from opening or closing. They can also detect whetherthe agent makes the ion channel more or less sensitive to voltagechanges.

FIG. 12 is a flowchart of another method of utilizing the cell assaysystems described above. In particular, FIG. 12 is a flowchart of amethod of identifying an unknown agent 1200. First, the impact ofvarious agents on voltage-gated ion-channels can be catalogued andstored in a database (steps 1202-1206). For example, each agent in alibrary of known agents processed according to a predetermined protocol.Each agent is introduced into an array of biological sample holdersholding one or more types of cells (step 1202). The cells are thenelectrically stimulated according to a predetermined protocol (step1204) and the resulting fluorescence of the optical ion sensors in thebiological sample holders is recorded and catalogued (step 1206). Thisrecord of fluorescence serves as a fingerprint for the agent. Toidentify an unknown agent (steps 1208-1212), the agent is subjected tothe same protocol used to obtain the fluorescence fingerprints usedpopulate the database (step 1208). The resultant fluorescence of theoptical ion sensors monitoring the effect of the agent on the cells inthe array is then compared with the fingerprints of known agents (step1210) to identify the unknown agent (step 1212).

Additional applications and protocols for analyzing ion-channel activityare described in U.S. Pat. No. 6,969,449, the entirety of which isincorporated by reference. Such protocols can be readily adapted for usewith the optical ion sensors and cell assay systems described herein.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The forgoingembodiments are therefore to be considered in all respects illustrative,rather than limiting of the invention.

1. A cell assay system comprising: a post having a distal end sized forintroduction into a biological sample holder; a plurality of electrodes,at least one of which is coupled to the post, for generating an electricfield between the electrodes when introduced into the biological sampleholder; a polymer-based optical ion sensor positioned proximate thedistal end of the post, wherein the optical ion sensor includes at leastone ionophore for selectively binding a predetermined ion, therebyaltering a pH of the optical ion sensor, and one pH-sensitivechromionophore for optically indicating the concentration thepredetermined ion based on the pH of the optical ion sensor and theresulting fluorescence of the chromionophore.
 2. The cell assay systemof claim 1, wherein the optical ion sensor is removably coupled to thepost.
 3. The cell assay system of claim 1, wherein the optical ionsensor is coupled to the biological sample holder.
 4. The cell assaysystem of claim 1, wherein the optical ion sensor is suspending in afluid in the biological sample holder.
 5. The cell assay system of claim1, wherein the optical ion sensor is located within a cell in thebiological sample holder.
 6. The cell assay system of claim 1, whereinthe optical ion sensor is disposed on a transparent surface positionedin the biological sample holder.
 7. The cell assay system of claim 1,wherein the optical ion sensor is located outside of a cell when thepost is introduced into the biological sample holder and a secondoptical ion sensor is located within a cell in the biological sampleholder.
 8. The cell assay system of claim 7, wherein the second opticalion sensor includes a different ionophore than the optical ion sensorfor indicating the concentration of two ions.
 9. The cell assay systemof claim 8, wherein the second optical ion sensor includes a differentchromionophore than the optical ion sensor.
 10. The cell assay system ofclaim 7, wherein the second optical ion sensor includes the sameionophore as the optical ion sensor.
 11. The cell assay system of claim1, comprising a controllable voltage source for generating a voltageacross the electrodes.
 12. The cell assay system of claim 1, wherein thecontrollable voltage source generates a voltage sufficient toelectroporate a cell in the biological sample holder.
 13. The cell assaysystem of claim 1, wherein the controllable voltage source generates avoltage sufficient to activate an ion channel in a cell in thebiological sample holder.
 14. The cell assays system of claim 1, whereinthe post comprises an agent introduction means for introducing an agentinto the biological sample holder.
 15. The cell assay system of claim14, wherein the agent introduction means comprises a hole in the post.16. The cell assay system of claim 14, wherein the agent introductionmeans comprises a pipette.
 17. The cell assay system of claim 14,wherein the agent introduction means comprises a electromechanical agentdispenser.
 18. The cell assay system of claim 17, wherein theelectromechanical agent dispenser comprises a solenoid.
 19. The cellassay system of claim 1, wherein the plurality of electrodes comprisestwo parallel electrodes coupled to opposing portions of the post. 20.The cell assay system of claim 1, wherein the plurality of electrodescomprises two coaxial electrodes.
 21. The cell assay system of claim 1,wherein at least one of the plurality of electrodes couples to thebiological sample holder.
 22. The cell assay system of claim 1, whereinat least one of the plurality of electrodes comprises a transparentconductor.
 23. The cell assay system of claim 1, wherein the pluralityof electrodes comprises first and second pairs of parallel electrodes,wherein the first pair of electrodes is aligned perpendicular to thesecond pair of electrodes.
 24. The cell assay system of claim 1,comprising a light sensor for measuring the fluorescence of the opticalion sensors.
 25. The cell assay system of claim 24, comprising acomputing device for measuring the output of the light sensor.
 26. Thecell assay system of claim 25, wherein the computing device comprises avoltage control module for causing varying voltages to be generatedacross the electrodes.
 27. The cell assay system of claim 25, whereinthe computing device comprises an analysis module for comparing theoutput of the light sensor at the various voltages.
 28. The cell assaysystem of claim 25, wherein the computing device comprises an agentintroduction control module for controlling the introduction of an agentinto the biological sample holder.
 29. The cell assay system of claim28, wherein the computer comprises an analysis module for comparing theoutput of the light sensor before an introduction of the agent into thebiological sample holder to the output of the light sensor after theintroduction of the agent into the biological sample holder.
 30. Thecell assay system of claim 1, comprising an excitation light source forexciting the optical ion sensor.
 31. A cell assay system, comprising: anarray of posts having distal ends spaced and sized for introduction intoan array of biological sample holders; a plurality of electrode sets,wherein at least one electrode of each electrode set is coupled to arespective post, and each electrode set is configured to generate anelectric field when introduced into a corresponding biological sampleholder in the array of biological sample holders; a plurality ofpolymer-based optical ion sensors positioned proximate the distal end ofthe respective posts in the array of posts, wherein the optical ionsensors include at least one ionophore for selectively binding apredetermined ion, thereby altering a pH of the optical ion sensor, andone pH-sensitive chromionophore for optically indicating theconcentration of the predetermined ion based on the pH of the opticalion sensor and the resulting fluorescence of the chromionophore.
 32. Thecell assay system of claim 31, wherein at least two of the optical ionsensors in the plurality of ion sensors include different ionophores toindicate the concentration of multiple predetermined ions correspondingto the different ionophores.
 33. The cell assay system of claim 32,wherein the at least two different optical ion sensors are proximate asingle post in the array of posts.
 34. The cell assay system of claim32, wherein the at least two different optical ion sensors are proximatedifferent posts in the array of posts.
 35. The cell assay system ofclaim 31, wherein the array of biological sample holders comprises oneof a 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, and a1534-well plate.
 36. The cell assay system of claim 31, comprising acomputing device including an agent introduction control module forcontrolling the introduction of an agent into at least one of thebiological sample holders.
 37. The cell assay system of claim 36,wherein the agent introduction control module controls the introductionof a plurality of agents into respective ones of the biological sampleholders in the array of biological sample holders.
 38. The cell assaysystem of claim 37, wherein the computing device comprises an analysismodule for comparing the fluorescence of the plurality of optical ionsensors before an introduction of at least one agent into at least oneof the respective biological sample holders to the fluorescence of thecorresponding plurality of optical ion sensors after the introduction ofthe at least one agent into the respective biological sample holders.39. The cell assay system of claim 31, comprising a robotics module forrobotically introducing the array of posts into the array of biologicalsample holders.
 40. The cell assay system of claim 39, wherein therobotics module is configured to introduce the array of posts into aplurality of arrays of biological sample holders in sequence.
 41. Amethod of conducting a biological assay comprising: introducing apolymer-based optical ion sensor into a biological sample holder,wherein the optical ion sensor includes at least one ionophore forselectively binding a predetermined ion, thereby altering a pH of theoptical ion sensor, and one pH-sensitive chromionophore for opticallyindicating the concentration of the predetermined ion based on the pH ofthe optical ion sensor and the resulting fluorescence of thechromionophore; generating an electric field across a cell in thebiological sample holder; measuring an output of a light sensormonitoring the a fluorescence of the optical ion sensor in response tothe generation of the electric field.
 42. The method of claim 41,comprising varying the electric field.
 43. The method of claim 41,comprising introducing an agent into the biological sample holder. 44.The method of claim 43, comprising detecting a change in the output ofthe light sensor in response to the introduction of the agent.
 45. Themethod of claim 44, comprising determining that the agent is toxic. 46.The method of claim 44, comprising detecting a nerve toxin.
 47. Themethod of claim 44, comprising detecting a heart toxin.
 48. The methodof claim 44, comprising determining that the agent is a candidate fortreating a condition.
 49. The method of claim 44, comprising comparingthe change in the output of the light sensor to the change of output ofthe light sensor caused by a plurality of known agents.
 50. The methodof claim 49, comprising identifying the agent.
 51. The method of claim41, wherein the optical ion sensor is coupled to a post, and whereinintroducing the optical ion sensor into the biological sample holdercomprises positioning the post in the biological sample holder.
 52. Themethod of claim 51, wherein the electric field is generated at least inpart by an electrode coupled to the post.
 53. The method of claim 41,wherein the optical ion sensor comprises an optical ion sensor particle,and wherein introducing the optical ion sensor into the biologicalsample holder comprises dispensing the optical ion sensor particle intoa fluid in the biological sample holder.
 54. The method of claim 53,comprising introducing the optical ion sensor into a cell located in thebiological sample holder.
 55. The method of claim 54, comprisingelectroporating the cell, thereby allowing the optical ion sensor toenter the cell.
 56. The method of claim 54, comprising applying acompound to the optical ion sensor to breach a vesicle formed by thecell around the optical ion sensor.
 57. The method of claim 41, whereinthe electric field is generated, at least in part, by an electrodecoupled to a post positioned in the biological sample holder.
 58. Themethod of claim 41, wherein introducing the optical ion sensor into thebiological sample holder comprises inserting the optical ion sensor intoa cavity formed in the post.
 59. The method of claim 41, comprisingforming a cell monolayer on the interior of the biological sampleholder.
 60. The method of claim 41, wherein introducing the optical ionsensor into a biological sample holder comprises introducing a firstoptical ion sensor into a cell in the biological sample holder andintroducing a second optical ion sensor into the biological sampleholder outside of the cell.
 61. A method of conducting a biologicalassay comprising: providing an array of biological sample holders;introducing an array of polymer-based optical ion sensors intobiological sample holders in the array of biological sample holders,wherein the optical ion sensors include at least one ionophore forselectively binding a predetermined ion, thereby altering a pH of theoptical ion sensor, and one pH-sensitive chromionophore for opticallyindicating the concentration of an ion corresponding to the ionophorebased on the pH of the optical ion sensor and the resulting fluorescenceof the chromionophore; generating electric fields across cells in thebiological sample holders in the array of biological sample holders;measuring an output of a light sensor monitoring the a fluorescence ofthe array of optical ion sensors in response to the electric fields. 62.The method of claim 61, comprising introducing a first agent into one ofthe biological sample holders in the array of biological sample holders.63. The method of claim 62, comprising introducing a second agent into asecond of the biological sample holders in the array of biologicalsample holders.
 64. The method of claim 63, detecting a change in theoutput of the light sensor resulting from the introduction of the firstand second agents.
 65. The method of claim 61, comprising, providing asecond array of biological sample holders and robotically introducingthe array of optical ion sensors into the second array of biologicalsample holders.